This invention relates to the inventive procedure for identifying xe2x80x9canti-virulencexe2x80x9d genes which are incompatible with virulence. This inventive principle is illustrated by the identification of the anti-virulence gene cadA. A further aspect of this invention is derived from this identification, because the gene products of these anti-virulence genes, or the compounds generated enzymatically by these gene products, are useful as pharmaceuticals. As exemplified by the compound generated by the product of the cadA gene (lysine decarboxylase), the invention relates to the use as a pharmaceutical of the diaminoalkanes and polyaminoalkanes in the treatment and prevention of pathogenic bacterial infections. Of particular use is cadaverine (1,5-diaminopentane).
As further support, and as a further embodiment, is the identification of nadA and nadB as anti-virulence genes, and the pharmaceutical use of the corresponding enzymatically generated compound, quinolinate.
The identification of a gene related to non-pathogenicity also permits the modification of pathogenic bacteria with the gene to thereby diminish pathogenicity. Thus, the invention includes modified pathogenic bacteria, including DNA constructs, vectors, plasmids, and organisms. This aspect of the invention permits the generation of vaccines that could protect against Shigella and other pathogenic bacteria such as enteroinvasive bacteria. The invention also encompasses delivery of DNA vaccines, where a gene of interest, or a fragment thereof, is delivered via these attenuated bacteria to the digestive epithelia without inducing diarrhea.
The invention further relates to an assay for pathogenicity by probing for cadA gene deletion in bacterial samples or cultures of unknown pathogenicity. Alternatively, pathogenicity may be determined by assaying for lysine decarboxylase activity as the protein product of this gene. The invention includes DNA constructs, fragments, vectors, plasmids and antibodies useful for constructing such pathogenicity assays.
Mankind exists in conjunction with a world of microorganisms whose number far outnumbers man""s. Some microorganisms are friendly co-habitants such as Escherichia coli, while others such as Shigella cause such dangerous maladies as bloody diarrhea (hemorrhagic colitis), hemolytic uremic syndrome, and dysentery.
Bacteria of the genus Shigella are gram negative enteric pathogens which are the causative agents of bacillary dysentery or shigellosis. Shigella infection accounts for a considerable fraction of acute diarrheal diseases worldwide and is an important public health problem in developing countries where bacillary dysentery remains a major cause of childhood mortality. The worldwide incidence of bacillary dysentery is estimated to exceed 200 million cases annually. About 5 million cases require hospitalization and about 650,000 persons die of shigellosis each year (Institute of Medicine (1986) The prospect for immunizing against Shigella spp., p. 329-337. In: New Vaccine Development: Establishing priorities. Vol. 2 Diseases of importance in developing countries. National Academy Press, Washington, D.C.). Shigellosis continues to be an important public health concern even in the United States with over 32,000 cases reported in 1995 (Centers for Disease Control and Prevention. 1995. Summary of notifiable diseases, United States, 1995. MMWR 44:1-3.). Of principal importance are foodborne outbreaks and outbreaks in institutional settings (day care centers, nursing homes, etc.) and on Indian reservations. The clinical presentation of shigellosis can range from a mild diarrhea to severe dysentery with frequent passage of bloody, mucoid, small volume stools. The disease is characterized by extensive damage to the colonic epithelial layer, cell death, ulceration and inflammation of the colon. While infections are usually self-limiting and do not spread from the lamina propria to the submucosa, shigellosis can be life-threatening in young or malnourished patients (DuPont, H. L. 1995. Shigella species (Bacillary dysentery), p. 2033-2039. In G. L. Mandell, et al. (eds.), Principles and Practice of Infectious Diseases. Churchhill Livingstone Inc., New York, N.Y.).
The primary means of human to human transmission of Shigella is by the fecal-oral route. Most cases of shigellosis are caused by the ingestion of fecally-contaminated food or water. In the case of foods, the major factor for contamination is the poor personal hygiene of food handlers. The low infectious dose of Shigella spp. presents a challenging problem. Volunteer studies showed that the ID50 (the infectious dose required to cause disease in 50% of the volunteers) of Shigella is as low as 200 shigellae although it has been reported that the ingestion of as few as 10 organisms is sufficient to cause disease (DuPont. H. L., et al. (1989) J. Infect Dis. 159:1126-1128). The low ID50 of Shigella accounts for its high communicability, particularly in impoverished and crowded populations. One consequence of this feature is that a contaminated food source has the potential to cause explosive outbreaks of dysentery with secondary cases likely to occur among close contacts of infected individuals. Thus, infected food handlers can contaminate food and spread infection among large numbers of individuals.
Maurelli and Lampel describe several examples of foodborne outbreaks of shigellosis (Maurelli and Lampel (1997) Shigella species, p. 216-227. In M. P. Doyle, et al. (eds.), Food Microbiology: Fundamentals and Frontiers. American Society for Microbiology Press, Washington, D.C.). Daycare workers and children attending day care facilities are placed at risk when a child infected with Shigella is present. The bacteria are shed in feces and the immature personal hygiene habits of very young children can easily lead to infection of other children as well as care providers (Mohle-Boetani, J. C., et al. (1995) Am. J. Pub. Health 85:812-816). With a low infectious dose required to cause disease coupled with oral transmission via fecally-contaminated food and water, it is not surprising that dysentery caused by Shigella spp. follows in the wake of many natural (earthquakes, floods, famine) and man-made disasters (war). Civil wars in Burundi and Rwanda led to massive movement of refugees. An outbreak of dysentery in a refugee camp in Rwanda in late 1993 affected more than 6,000 people (attack rate greater than 32%), mostly children under five years old (Paquet, C., et al. (1995) Une epidemie de dysenteriae à Shigella dysenteriae type 1 dans un camp de refugies au Rwanda, Sautxc3xa9 5:181-184). In August, 1994, more than 15,500 cases of bloody diarrhea were reported from three refugee camps in Zaire (Centers for Disease Control and Prevention. (1996) Morbidity and mortality surveillance in Rwandan refugeesxe2x80x94Burundi and Zaire, 1994. MMWR 45:104-107). All of these factors are exacerbated by the fact that shigellae are becoming increasingly resistant to most antimicrobial agents commonly used in the treatment of diarrheal diseases (Centers for Disease Control and Prevention. (1994) Addressing emerging infectious disease threats: A prevention strategy for the United States. U.S. Department of Health and Human Services, Public Health Service, Atlanta, Ga.).
There exists no effective vaccine against shigellosis. Previous attempts to develop a vaccine against enteropathogenic bacteria have suffered from a failure to engender immunogenicity without also generating diarrhea, or from requiring multiple doses as well as boosters of high numbers of viable bacteria. Thus, there is an unsatisfied need in the art for a vaccine against shigellosis. Such a vaccine may be a bacteria which is enteroinvasive and thereby capable of delivering an immunogen to a host, but which is not reactogenic or diarrheic in the host (Sizemore, D. R., et al. (1995) Science 270:299-302).
Shigella are also interesting in that they share significant homology with E. coli, a generally benign bacteria. Indeed, the four species of Shigella are so closely related to E. coli that all of these bacteria could be considered members of a single species. They share greater than 90% homology by DNAxe2x80x94DNA reassociation analysis (Brenner, D. J., et al. (1969) J. Bacteriol. 98, 637-650) and display colinearity of their chromosomes such that gene transfer by conjugation and transduction and formation of recombinants between Shigella and E. coli occur with high efficiency (Formal, S. B., et al. (1970) Infect. Immun., 1, 179-287). Nevertheless, Shigella spp. are serious pathogens that cause bacillary dysentery, whereas E. coli (with the exception of certain pathogenic clones) are commensals of the human intestine.
Given the similarity between pathogens, such as the toxic Shigella, and commensals, such as the benign E. coli, there exists a need in the art for a simple and reliable test to distinguish between these closely related microorganisms. In addition, there exists a need for probes to identify receptors associated with these bacteria and their toxins for the ultimate goal of providing information on the evolutionary basis of pathogenicity. Knowledge of receptor characteristics would facilitate rational vaccine design wherein the naturally invasive nature of these bacteria may be exploited in positive ways. Furthermore, because of the severity and widespread nature of pathogenic bacterial infections, there exists a need in the art for a rational approach to this analysis of virulence and pathogenesis, and also for a rational approach to the discovery of new pharmaceuticals for treatment of pathogenic bacterial infections.
Thus, there is a need in the art for an effective method of preventing or treating diseases caused by pathogenic organisms such as Shigella as well as ways of distinguishing such pathogenic organisms from nonpathogenic organisms.
The present invention satisfies these needs by describing the correlation between pathogenicity and a known gene, cad, which produces the enzyme lysine decarboxylase (LDC) and the applications of this correlation. In many wild-type bacteria, presence of this gene is correlated with lack of pathogenicity. Based on this observation, we have identified a new inhibitor of the enterotoxins produced by pathogenic bacteria: the enzymatically generated product of LDCxe2x80x94cadaverine. Cadaverine is a diaminoalkyl compound, only one of a large related class of such compounds, which provides promising therapeutics for the treatment and prevention of pathogenic bacterial infections. Additionally, we have discovered that expression of this gene is correlated only with decreased diarrheic ability, while retaining invasive action, an effect which is invaluable for designing DNA vaccine delivery vehicles and vaccines against enteroinvasive bacteria.
This discovery marks an important step in the rational analysis of virulence, and in the rational search for new treatment strategies and pharmaceuticals. Although the evolution of bacterial pathogens from non-pathogenic ancestors has been thought to proceed through the acquisition of virulence genes, we have discovered that the loss of xe2x80x9canti-virulencexe2x80x9d genes causes genomic black holes and results in a pathogenic phenotype.
This discovery enables the discovery of new pharmaceuticals by merely comparing the genomes of a pair of closely related virulent/avirulent species, identifying missing xe2x80x9canti-virulencexe2x80x9d genes in the virulent strain, and testing the product(s) of this missing gene (both the protein, and any enzymatically generated compounds) for inhibitory effects on the invasive bacteria, or prophylactic effects on the disease condition. This approach has been successfully demonstrated for Shigella flexneri, where the xe2x80x9canti-virulencexe2x80x9d gene is cadA, and the new pharmaceutical is cadaverine, but it is equally applicable to other pathogenic bacteria. Initial results also have identified two more anti-virulence genes, nadA and nadB, and their corresponding enzymatically produced compound, quinolinate.